Single-use Stationary Bioreactors and Mixing Vessels

ABSTRACT

Stationary bioreactors and mixing vessels fitted with single-use flexible bags utilizing wave hydrodynamic principle are described for use in every type of biological process and products.

BACKGROUND

The use of single-use plastic bags in the biological manufacturing,blood-banking and other tissue and cell culture processing is fastbecoming the most desirable method as their use obviates the need toclean the vessels and validate them for future operations; withincreased demand by the regulatory authorities to provide cleaningvalidation proof, particularly those of freedom from viruses, it isanticipated that soon, because of higher cost of validation, most of thebiological reaction and storage processes will be carried out insingle-use flexible plastic bags. A large number of commercial suppliersare currently marketing these single-use flexible plastic bag systemsfor biological manufacturing as well as storage of biological fluidssuch as blood bags; of prime interest to this invention are the mixingsystems offered by GE Healthcare as the Wave Bag system wherein atwo-dimensional bag is affixed to a rocking platform to mix the contentsto produce biological compositions such as culture media and when fittedwith appropriate systems, to induce bioreaction in the presence ofnecessary nutrients and biocatalysts. The U.S. Pat. No. 6,190,913 toVijay Singh describes the details of the Wave system currently marketedby GE Healthcare. The GE Healthcare Wave Cellbag is intended to be usedwith a gentle motion and as a result it has been recommended by themanufacturer not to use the system for reactions requiring more vigorousmixing such as required in the fermentation using bacteria or otherorganisms or cells severely limiting the use of the Wave Cellbag andother similar apparatus in the commercial manufacture of biologicalproducts.

Another competing technology utilizes flexible bags attached withstirrers and other implements similar to what is used in the traditionalstainless steel fermentors or mixers. In some instances, mixing isachieved by a variety of aeration mechanisms along with mechanicalmixing but until now no invention has described a stationary system thatwill induce mixing inside flexible bags such that the size of the bagcan be infinitely variable and the nature and intensity of mixingadjustable to the need of the bioreaction.

SUMMARY OF INVENTION

There are two types of single-use bioreactors available today; one thatdo not have retaining walls such as the GE Wave system and those thathave retaining walls such as the Xcellerex, Sartorius, Millipore andothers. The GE bioreactor can only manage media volume of up to 500 L ina 1000 L bag before the physical limit of bag strength or manageabilityis reached; for the hard-walled support systems, the limitation ofproviding mechanical stirring devices at larger scale makes themimpractical; the largest size of single-use bioreactors available todayis offered by Xcellerex at 2000 L at a cost of over two million dollarsand with recurring costs comparable to those incurred when using thetraditional stainless steel bioreactors.

Despite the obvious savings in capital expenditures and labor costs andimproved facility operations, it is unlikely that single-use, single-usetechnologies will entirely supplant or eliminate the use ofstainless-steel systems in bio-manufacturing mainly because of sizelimitations and physical constraints, in the opinion of the industryleaders. There is therefore an unmet need to invent an apparatus thatwould allow unlimited substitution of the stainless steel systems bysingle-use flexible bag systems.

A variety of vessels and methods have been developed over the years tocarry out the fermentation of microorganisms, particularly bacteria andyeast, on a commercial scale. Stainless steel fermentation vessels ofhundreds of thousands of liters are not uncommon, with the fermentationmethods including batch, fed-batch, and continuous or semi-continuousperfusion. The cells within these vessels are desirably kept insuspension, typically by rotating stirring blades located within thevessel, with gas exchange facilitated by the injection of air, oxygen orcarbon dioxide into the vessel. There are several drawbacks to thisdesign. One is the introduction of shearing forces through the stirringblades and the cavitations of miniscule air bubbles, both beingdetrimental to more sensitive cell types or organisms. Also, thesevessels should be rigorously cleaned between production runs to preventcross-contamination, the latter being time consuming and requiringvalidation for individual cultures. Furthermore, the cost of stirredfermentors is relatively high on a volume basis, and thus thesefermentors are commonly used over long periods of time. This, however,increases the risk of undesirable infection of mechanical failures.Perhaps most significantly, the optimization of culture conditions forstirred fermentors in a small scale cannot be transferred in a linearway to commercial scale production. For example, the fluid dynamics,aeration, foaming and cell growth properties change when the scaleincreases. In addition, for more delicate cell types or organisms, alarge-scale stirred fermentation vessel is not a viable device, evenwhen more subtle stirring techniques such as airlift fermentors areused.

These drawbacks have led to the development of single-use fermentors.Examples of such single-use fermentors are systems based on waveagitation. See, e.g., U.S. Pat. No. 6,544,788; PCT Publication WO00/66706. This type of fermentor may be used to culture relativelysensitive cells such as CHO cells (e.g., Pierce, Bioprocessing J. 3:51-56 (2004)), hybridoma cells (e.g., Ling et al., Biotech. Prog., 19:158-162 (2003)), insect cells (e.g., Weber et al., Cytotech. 38: 77-85(2002)) and anchorage-dependent cells (e.g., Singh, Cytotech. 30:149-158 (1999)) in a single single-use container. Such single-use unitsare relatively cheap, decrease the risk of infection because of theirsingle use and require no internal stirring parts as the rockingplatform upon which these containers reside during use induces wave-likeforms in the internal liquid, which facilitates gas exchange. However,this principle cannot be expanded to the size of hundreds of thousandsof liters (such as the industrial fermentors) but are currentlyavailable from 1 liter to 500 liters (total volume of the single-usebag, available from Wave Biotechnology AG, Switzerland; Wave BiotechInc., USA). Moreover, the hydrodynamics for each size of single-use bagwill differ as a result of differences in depth and height. Therefore,the use of these single-use bags requires optimization and re-validationof each step in an up scaling process. Thermo Fisher Hyclone was thefirst to introduce the single-use, single-use bioreactor concept andoffers various sizes of vertical, cylindrical bioreactor bags that aredesigned to fit into a stainless outer vessel support with a heatingjacket and a single-use mixing mechanism. Aeration, mixing, andbiosensor probes are introduced into the bag through welded elastomersleeves.

Sartorius Stedim and GE Healthcare/Wave manufacture bioreactor bags in aflat configuration agitated by the motion of a rocker platform. Like theThermo Fisher Hyclone configuration, mixing, aeration, and probes areinserted into the Wave and Sartorius Stedim through welded bag ports.

ATMI Life Sciences recently introduced a rectangular-shaped single-usebioreactor bag with a top-mounted agitator rod.

Xcellerex manufactures a single-use bioreactor bag that fits into asupport shell with a magnetically coupled bottom agitation system(stirred-tank) for mixing.

Although bioreactor systems and related processes are known,improvements to such systems and processes would be useful in thepreparation of a variety of products produced from a biological source.A similar situation arises when flexible bags are used for the purposeof mixing the contents such as in the preparation of culture media orbuffers; the long-time use of flexible plastic bags is well known in theblood banking industry where often the need arises for the mixing ofcontents to reduce the degradation of the contents. Yet anothersignificant application that pertains the mixing function arises in themanufacture of recombinant proteins wherein the protein is allowed tounfold in a very dilute solution at low temperature. To obtain theoptimal yield upon refolding the solution must be gently shaken duringthe refolding period. All of these operations are dependent on theefficiency of the mixing methods.

The technology for mixing and aeration also forms the core of theengineering design that allows biological reactions to take place invessels called fermentors or bioreactors. Hard-walled stainless steelreactors are available in all sizes from a few liters to thousands ofliters and are the mainstay of the manufacturing of biotechnologyproducts. However, the flexible plastic bag use (without retainingwalls) is severely limited by the size of the batch as it becomesmechanically unmanageable to provide a suitable mixing cycle in thecontainer because of the large size and weight on the platform thatimparts the motion to the flexible bag. Any type of mechanical motionwhether it is rocking, orbital or reciprocal requires a tremendousamount of energy to move the flexible plastic container filled withmedia.

The requirement of high agitation of liquid media in the fermentation ofmicroorganisms is one reason why flexible containers should be used forgrowing microorganisms. The difficulty arises in shaking vigorously theentire container that may weight hundreds of even thousands of poundsproducing stress on the walls of the container besides needing verylarge mechanical systems to operate these shaking protocols. The instantinvention resolves this problem by keeping the bag stationary on a flatsurface and only creating motion inside the bag by a squeeze motion thatproduces many different types of mixing, from very gentle to veryvigorous, the latter being a requirement of growing microorganisms andthus allowing the use of flexible containers for the purpose of growingmicroorganisms. The instant invention capitalizes on a well-knownphenomenon of liquid wave propagation wherein a movement created in onepart of a continuous liquid media travels and spreads to all other partsof liquid media.

To remove the limitation of the size of the mixing or bioreacting bag insingle-use environment, the instant invention eliminates the need forproviding any motion at all to the bag. Instead, the mixing motion,whether in the form of a gentle wave or intense turbulence, is inducedinside the bag by applying compression at one end of the bag filled withliquids and by applying such action repeatedly in a cyclical manner, awave motion is created that allows gentle mixing of components in thesinge-use containers; however the amplitude and frequency of edgecompression can easily lead to extreme turbulent motion inside the bag.

Thus a preferred embodiment of this invention is a method of mixingcontents in flexible plastic containers by applying compression forcealong one edge of the bag and then reversing the compression on theother side to create a wave motion.

In another preferred embodiment of the instant invention the compressionat the edge of the bag is provided at all four edges in a cycle thatproduces vortex inside the bag.

In another preferred embodiment the instant invention provides a methodof mixing and bioreacting any size of flexible containers.

In another preferred embodiment the instant invention provides a methodfor fermentation and bioreaction for growth of cells and microorganisms.

In another preferred embodiment the instant invention provide a methodfor mixing of fluids in flexible bags such that the contents are kepthomogenous.

In another preferred embodiment the instant invention provides a methodfor refolding of proteins in recombinant manufacturing of drugs whereinrefolding media is stored at low temperature and mixed gently forextended time.

In another preferred embodiment the instant invention provides a methodof extending the storage life of biological products whereby gentlemixing is required to prevent degradation.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is the angle view of the bioreactor showing the flaps, themotorized drive to move them to affect hydrodynamic motion inside theflexible bag.

FIG. 2 is the angle view of the bioreactor as described in FIG. 1 andincluding interconnecting rods to move opposite flaps insynchronization.

DETAILS OF INVENTION

The instant invention is based on several essential componentscomponents. First, there is a two or three-dimensional flexible bag thatcan contain various volumes of liquid products or media. The flexiblebag may additionally contain other features as required for the aerationof media, exhaust of gases, introduction of culture media, filtrationand removal of media or any other such feature that may be required toachieve the intent of the process. For example, one such instant willthe Bio Bag supplied by Wave Systems that is available in sizes rangingfrom 5 L to 1000 L. This bag can be used without any modification in theinstant invention. However, the most expensive part of the Wave Systeminvolves a rocking platform that also limits the size of liquid producthandled to about 500 L since beyond this size, it becomes mechanicallyprohibitive and thus restricts the applications of flexible bags inbio-manufacturing. Similarly, flexible bags of any design, shape or formcan be used in the instant invention without further modification.However, in all such instances the mixing mechanism is replaced with themethod described in the instant invention.

Secondly, a support surface is provided for the flexible bags of a sizesuch that the flexible bags would fit within the dimensions of thesupport surface.

Third, the support surface is provided with flaps that also act as sidesupport in the form of short walls of a size such that when the flexiblebag is fully inflated, the walls will prevent it from tipping or fallingoutside.

Fourth, the flaps described in the feature Three are attached to thesupporting surface by a hinge that will allow movement of the flaps inboth directions; the flaps are pulled down when loading the supportingsurface and then raised after the loading of the flexible bag.

Fifth, the flaps can be moved horizontally to bring them closer to theflexible containers to provide better contact and efficiency ofoperation.

Sixth, the flaps area connected to diagonally opposite flaps by amechanical means such that the movement of one flap also forces themovement of the opposing flap in opposite direction.

Seventh, the moveable flaps are provided with at least one means ofmovement in the direction of the bag and using a motorized cam or linearactuator that can be adjusted for amplitude and frequency.

Eight, the instant invention is operated by pushing the flaps either inone dimension of in both dimensions in a reciprocal motion such that theflexible bag is poked by the movement of flaps and this action isfollowed by a similar application of force on the opposite of theflexible bag; the repeated application of this force thus creates arepeatable motion within the liquid contained in the flexible bag.

Nine, the instant invention optionally provides means of keeping thesupport surface hot or cold as may be required by the process byintroduction of means of heating or cooling attached to the underside ofthe support surface.

As a preferred embodiment FIG. 1 describes the construction theapparatus. A flat support surface 1 is connected to flaps 2 on all foursides by hinges; the flaps are attached to a linear actuator 3 throughits push and pull rod 4 such that upon activating the actuator, the flap2 is moved vertically in either direction. The flat surface 1 isprovided with means of heating or cooling it as feature 5 and adjustingthe position of flaps 2 on the adjustment rail 6 since the size offlexible bag may change requiring moving the flaps closer to the bag;the entire apparatus rests on legs 7 and contained inside the apparatusis a flexible bag 8.

FIG. 2 describes the same features as shown in FIG. 1 and additionallyincludes inter-connecting rods 9, which when installed allow synchronousmovement of opposite flaps using only linear actuator 3 for theoperation of two opposite flaps. In a typical operation, first the flaps2 on one of each of the long and short side of the flat surface 1 areopened down to allow easy access to a flexible container 8 filled withculture media and attached to other systems to aerate the culture willbe placed on surface 1 and the surface 1 heated or cooled by operatingfeature 5, which is likely to be a block through which either cold orhot water is passed or contains a separate resistance type heater forheating surface 1. Now the flaps 2 are brought in close proximity to theflexible bag by adjusting the nuts on railing 6. If synchronizedmovement is required, the flaps 2 are interconnected with opposite flapsby means of connecting rods 9, which are also adjustable in size. Nowonce the assembly is complete, the apparatus is operated by switching onthe power to the linear actuator 3, which pushes flaps to a fixeddownward position and with frequency adjusted by the control of linearactuator 3. Once the reaction process is complete, the linear actuator 3is stopped, the flaps 2 folded down and the flexible bag 8 eitherremoved from the surface 1 with culture media or the culture media isfirst removed from the bag by a peristaltic pump through a tube insertedinside the bag (not shown).

In another embodiment, the flexible bag placed on support surface 1contains a protein solution undergoing refolding, when all operationsdescribed above will be applied except that the movement of flaps willbe slow and the surface 1 kept cold to a temperature of about 8 C-10 C.

In another embodiment, the flexible bag placed on support surface 1 isused to mix culture media or buffer wherein materials will be introducedin the bag, which is then allowed to mix the ingredients throughmovement of fluid, with moderately agitation.

In another embodiment, the contents of flexible bag on support surface 1are kept mixed through the action of flaps 2 for prolonged period oftime during storage of biological products.

1. An apparatus for mixing or agitating materials into final productcomprising: a flexible container with at least four edges, a topsurface, a bottom surface and containing said materials; a flatsupporting means with four sides, a top surface, a bottom surface and ofsufficient length and width to completely contain and hold said flexiblecontainer; at least one moveable flap affixed vertically to said topsurface of said supporting means; a means of adjusting the position ofsaid moveable flap; a mechanical means of moving said moveable flap ineither direction; an electronic or mechanical means of controlling theamplitude and the frequency of the movement of said moveable flap. 2.The apparatus of claim 1 in which a plurality of flaps is attachedvertically to said top surface of said supporting means providing atleast one flap along each of said sides of said supporting means.
 3. Amethod of inducing a wave-type mixing motion inside said flexiblecontainer by moving at least one of said flaps in the direction of saidflexible container with sufficient force to compress said top surface ofsaid flexible container along its said edge and then reversing thedirection of movement of said flap and repeating the operationperiodically.
 4. A method of inducing a reciprocal mixing motion insidesaid flexible container by moving two of said flaps attached to theopposing said sides of said supporting means in the direction of saidflexible container alternately with sufficient force to compress saidtop surface of said flexible container along its said edges and thenreversing the direction of movement of said flaps and repeating theoperation periodically.
 5. A method of inducing an orbital mixing motioninside said flexible container by moving all four of said moveable flapsin the direction of said flexible container sequentially with sufficientforce to compress said top surface of said flexible container along itssaid edges and then reversing the direction of movement of said flapsand repeating the operation periodically.
 6. A method of inducing avertical mixing motion inside said flexible container by moving at leasttwo of said moveable flaps attached to said opposing sides of saidsupporting means in the direction of said flexible containersimultaneously with sufficient force to compress said top surface ofsaid flexible container along its said edges and then reversing thedirection of the movement of said flap and repeating the operationperiodically.
 7. The apparatus of claim 1 in which said supporting meansis additionally provided with means of heating or cooling saidsupporting means to a desired temperature comprising at least one heatexchange means with circulating hot or cold liquids attached to saidbottom surface of said supporting means; at least one means ofmonitoring the temperature comprising at least one thermocouple attachedto said supporting means or said flexible container; at least one meansof adjusting said temperature of said supporting surface or saidflexible container by changing the flow rate of said circulating hot orcold liquids through said heat exchange means.
 8. The apparatus of claim1 in which said supporting means is additionally provided with means ofheating said supporting means to a desired temperature comprising atleast one electrical resistance heater attached to said bottom surfaceof said supporting means; at least one means of monitoring thetemperature comprising at least one thermocouple attached to saidsupporting means or said flexible container; at least one means ofadjusting the said temperature of said support means or said flexiblecontainer by adjusting the heat intensity of said electric resistanceheater.
 9. A method of growing microorganisms, yeasts, plant cells,baculoviruses, and animal cells comprising of introducing a growth mediainto said flexible container; introducing sufficient starter quantitiesof said microorganisms, yeasts, plant cells, baculoviruses, or animalcells in said flexible container; providing means of aeration of saidgrowth media in said flexible container; providing the required heatingor cooling of said culture media; operating said flaps to induce mixingor agitation of media; removing said growth media upon completion of thegrowth of said microorganisms, yeasts, plant cells, baculoviruses, oranimal cells reaction for further processing.
 10. A method of mixingchemical and biological products comprising of introducing a solvent insaid flexible container; introducing chemical or biological componentsinto said flexible container; operating said flaps to induce mixing oragitation of said solvent to form a solution; removing said solutionfrom said flexible container for further processing.
 11. A method ofrefolding proteins dissolved or suspended in a chemical buffercomprising of introducing said buffer solution of proteins in saidflexible container; applying sufficient cooling to said supporting meansas required; operating said flaps to induce gentle agitation action;removing said buffer solution after completion of process for furtherprocessing.
 12. A method of storing biological or chemical productscomprising of introducing said products in said flexible container;applying sufficient cooling to achieve optimal temperature for stablestorage of said biological or chemical products; optionally applyinggentle mixing motion to keep said chemical or biological productshomogenous.